Substituted imidazo[1,2-a]pyrazines as luciferase substrates

ABSTRACT

Described are substituted imidazo[1,2-a]pyrazine compounds, which are coelenterazine analogues, kits comprising the compounds, and methods of using the compounds for the detection of luminescence in luciferase-based assays. Also described are methods from making the compounds, such as a method using aminopyrazine acetophosphonates as synthesis intermediates.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/887,735, filed on Feb. 2, 2018, which is a divisional of U.S. patentapplication Ser. No. 15/431,961, filed on Feb. 14, 2017, now U.S. Pat.No. 9,924,073, which claims priority to U.S. Provisional PatentApplication No. 62/295,363, filed on Feb. 15, 2016, the entire contentsof each of which are incorporated herein by reference.

TECHNICAL FIELD

The present disclosure relates to coelenterazine analogues, methods formaking coelenterazine analogues, and methods of using coelenterazineanalogues in luciferase-based assays.

BACKGROUND

Bioluminescent assays are used extensively in the investigation ofcellular physiology, especially processes associated with geneexpression. In particular, luciferase reporter enzymes are quitevaluable tools in this field, and, to date, there has been intenseprotein engineering to obtain small and environmentally insensitiveluciferases that may be useful in bioluminescent assays. There exist anumber of efficient luciferase reporters enabling whole-cell biosensormeasurements, drug discovery through high-throughput screening, and invivo imaging, which also permits the study of protein-proteininteractions in living cells, apoptosis, and cell viability. Luciferasesthat use coelenterazine and coelenterazine analogues as substrates areamong the most widely used systems due to their brightness andacceptance in whole cell applications.

SUMMARY OF THE INVENTION

Many known coelenterazine analogues have deficiencies, which limit theireffectiveness as luciferase substrates and usefulness inluciferase-based luminescence assays. These deficiencies include celltoxicity, light sensitivity, thermodynamic instability, low aqueoussolubility, and low cell permeability. Accordingly, there exists a needfor coelenterazine analogues with improved properties and methods forsynthesizing the analogues.

In one aspect, disclosed are compounds of formula (I),

or tautomers, or pharmaceutically acceptable salts thereof, wherein R¹is alkyl, alkenyl, alkynyl, aryl, bicyclic aryl, tricyclic aryl,heteroaryl, bicyclic heteroaryl, tricyclic heteroaryl, heterocycle,cycloalkyl, heteroarylcarbonyl, or phosphonate; and q is 0-2; whereinsaid alkyl, alkenyl, alkynyl, aryl, bicyclic aryl, tricyclic aryl,heteroaryl, bicyclic heteroaryl, tricyclic heteroaryl, heterocycle,cycloalkyl, heteroarylcarbonyl, and phosphonate, at each occurrence, areindependently substituted or unsubstituted.

Also disclosed are methods of making the compounds, kits comprising thecompounds, and methods of using the compounds as luciferase substratesin luciferase-based luminescence assays.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting cellular bioluminescent activity ofexemplary compounds in HEK293 cells in comparison to coelenterazine.

FIG. 2 is a graph depicting the rank ordering of compounds of formula(I) by signal strength relative to coelenterazine.

FIG. 3 and FIG. 4 are graphs depicting results of a cell viability assayemploying exemplary compounds in HeLa cells.

FIG. 5 and FIG. 6 are graphs depicting results of a cell viability assayemploying exemplary compounds in HEK293 cells.

DETAILED DESCRIPTION

Disclosed herein are coelenterazine analogues. The coelenterazineanalogues can be compounds of formula (I) and useful substrates ofproteins that utilize coelenterazine to produce luminescence, including,but not limited to, luciferases and photoproteins found in variousmarine organisms such as cnidarians (e.g., Renilla luciferase),jellyfish (e.g., aequorin from the Aequorea jellyfish) and decapodsluciferases (e.g., luciferase complex of Oplophorus gracilirostris). Incomparison to coelenterazine, compounds of formula (I) may have at leastone of improved water solubility, improved stability, improved cellpermeability, increased biocompatibility with cells, reducedautoluminescence, and reduced toxicity.

Also disclosed herein are methods of making coelenterazine analogues[compounds of formula (I)]. Disclosed are three robust and versatileapproaches towards the preparation of coelenterazine analogues. Themethods allow for the preparation of analogues that could not have beenprepared using existing synthetic methods. The described methodologyenables access to a variety of substituents at the R¹ position and canbe performed under mild conditions utilizing a wide variety of readilyavailable starting materials. The disclosed synthetic methodologyunexpectedly provides a variety of new applications and advancements inbioluminescence technology based on coelenterazine analogues.

1. DEFINITIONS

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. In case of conflict, the present document, includingdefinitions, will control. Preferred methods and materials are describedbelow, although methods and materials similar or equivalent to thosedescribed herein can be used in practice or testing of the presentinvention. All publications, patent applications, patents and otherreferences mentioned herein are incorporated by reference in theirentirety. The materials, methods, and examples disclosed herein areillustrative only and not intended to be limiting.

The terms “comprise(s),” “include(s),” “having,” “has,” “can,”“contain(s),” and variants thereof, as used herein, are intended to beopen-ended transitional phrases, terms, or words that do not precludethe possibility of additional acts or structures. The singular forms“a,” “an” and “the” include plural references unless the context clearlydictates otherwise. The present disclosure also contemplates otherembodiments “comprising,” “consisting of” and “consisting essentiallyof,” the embodiments or elements presented herein, whether explicitlyset forth or not.

The modifier “about” used in connection with a quantity is inclusive ofthe stated value and has the meaning dictated by the context (forexample, it includes at least the degree of error associated with themeasurement of the particular quantity). The modifier “about” shouldalso be considered as disclosing the range defined by the absolutevalues of the two endpoints. For example, the expression “from about 2to about 4” also discloses the range “from 2 to 4.” The term “about” mayrefer to plus or minus 10% of the indicated number. For example, “about10%” may indicate a range of 9% to 11%, and “about 1” may mean from0.9-1.1. Other meanings of “about” may be apparent from the context,such as rounding off, so, for example “about 1” may also mean from 0.5to 1.4.

Definitions of specific functional groups and chemical terms aredescribed in more detail below. For purposes of this disclosure, thechemical elements are identified in accordance with the Periodic Tableof the Elements, CAS version, Handbook of Chemistry and Physics, 75^(th)Ed., inside cover, and specific functional groups are generally definedas described therein. Additionally, general principles of organicchemistry, as well as specific functional moieties and reactivity, aredescribed in Organic Chemistry, Thomas Sorrell, University ScienceBooks, Sausalito, 1999; Smith and March March's Advanced OrganicChemistry, 5^(th) Edition, John Wiley & Sons, Inc., New York, 2001;Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., NewYork, 1989; Carruthers, Some Modern Methods of Organic Synthesis, 3^(rd)Edition, Cambridge University Press, Cambridge, 1987; the entirecontents of each of which are incorporated herein by reference.

The term “alkoxy” as used herein, refers to an alkyl group, as definedherein, appended to the parent molecular moiety through an oxygen atom.Representative examples of alkoxy include, but are not limited to,methoxy, ethoxy, propoxy, 2-propoxy, butoxy and tert-butoxy.

The term “alkyl” as used herein, means a straight or branched, saturatedhydrocarbon chain containing from 1 to 10 carbon atoms. The term “loweralkyl” or “C₁-C₆-alkyl” means a straight or branched chain hydrocarboncontaining from 1 to 6 carbon atoms. The term “C₁-C₃-alkyl” means astraight or branched chain hydrocarbon containing from 1 to 3 carbonatoms. Representative examples of alkyl include, but are not limited to,methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl,tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl,2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, andn-decyl.

The term “alkenyl” as used herein, means a hydrocarbon chain containingfrom 2 to 10 carbon atoms with at least one carbon-carbon double bond.The alkenyl group may be substituted or unsubstituted. For example, thealkenyl group may be substituted with an aryl group, such as a phenyl.

The term “alkynyl” as used herein, means a hydrocarbon chain containingfrom 2 to 10 carbon atoms with at least one carbon-carbon triple bond.The alkynyl group may be substituted or unsubstituted. For example, thealkynyl group may be substituted with an aryl group, such as a phenyl.

The term “alkoxyalkyl” as used herein, refers to an alkoxy group, asdefined herein, appended to the parent molecular moiety through an alkylgroup, as defined herein.

The term “alkylene”, as used herein, refers to a divalent group derivedfrom a straight or branched chain hydrocarbon of 1 to 10 carbon atoms,for example, of 2 to 5 carbon atoms. Representative examples of alkyleneinclude, but are not limited to, —CH₂CH₂—, —CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂—,and —CH₂CH₂CH₂CH₂CH₂—.

The term “aryl” as used herein, refers to a phenyl group, or bicyclicaryl or tricyclic aryl fused ring systems. Bicyclic fused ring systemsare exemplified by a phenyl group appended to the parent molecularmoiety and fused to a phenyl group. Tricyclic fused ring systems areexemplified by a phenyl group appended to the parent molecular moietyand fused to two other phenyl groups. Representative examples ofbicyclic aryls include, but are not limited to, naphthyl. Representativeexamples of tricyclic aryls include, but are not limited to,anthracenyl. The monocyclic, bicyclic, and tricyclic aryls are connectedto the parent molecular moiety through any carbon atom contained withinthe rings, and can be unsubstituted or substituted.

The term “cycloalkyl” as used herein, refers to a carbocyclic ringsystem containing three to ten carbon atoms, zero heteroatoms and zerodouble bonds. Representative examples of cycloalkyl include, but are notlimited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, cyclooctyl, cyclononyl and cyclodecyl.

The term “cycloalkenyl” as used herein, means a non-aromatic monocyclicor multicyclic ring system containing at least one carbon-carbon doublebond and preferably having from 5-10 carbon atoms per ring. Exemplarymonocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl orcycloheptenyl.

The term “fluoroalkyl” as used herein, means an alkyl group, as definedherein, in which one, two, three, four, five, six, seven or eighthydrogen atoms are replaced by fluorine. Representative examples offluoroalkyl include, but are not limited to, 2-fluoroethyl,2,2,2-trifluoroethyl, trifluoromethyl, difluoromethyl, pentafluoroethyl,and trifluoropropyl such as 3,3,3-trifluoropropyl.

The term “alkoxyfluoroalkyl” as used herein, refers to an alkoxy group,as defined herein, appended to the parent molecular moiety through afluoroalkyl group, as defined herein.

The term “fluoroalkoxy” as used herein, means at least one fluoroalkylgroup, as defined herein, is appended to the parent molecular moietythrough an oxygen atom. Representative examples of fluoroalkyloxyinclude, but are not limited to, difluoromethoxy, trifluoromethoxy and2,2,2-trifluoroethoxy.

The term “halogen” or “halo” as used herein, means Cl, Br, I, or F.

The term “haloalkyl” as used herein, means an alkyl group, as definedherein, in which one, two, three, four, five, six, seven or eighthydrogen atoms are replaced by a halogen.

The term “haloalkoxy” as used herein, means at least one haloalkylgroup, as defined herein, is appended to the parent molecular moietythrough an oxygen atom.

The term “heteroalkyl” as used herein, means an alkyl group, as definedherein, in which one or more of the carbon atoms has been replaced by aheteroatom selected from S, Si, O, P and N. The heteroatom may beoxidized. Representative examples of heteroalkyls include, but are notlimited to, alkyl ethers, secondary and tertiary alkyl amines, amides,and alkyl sulfides.

The term “heteroaryl” as used herein, refers to an aromatic monocyclicring or an aromatic bicyclic ring system or an aromatic tricyclic ringsystem. The aromatic monocyclic rings are five or six membered ringscontaining at least one heteroatom independently selected from the groupconsisting of N, O and S (e.g. 1, 2, 3, or 4 heteroatoms independentlyselected from O, S, and N). The five membered aromatic monocyclic ringshave two double bonds and the six membered six membered aromaticmonocyclic rings have three double bonds. The bicyclic heteroaryl groupsare exemplified by a monocyclic heteroaryl ring appended to the parentmolecular moiety and fused to a monocyclic cycloalkyl group, as definedherein, a monocyclic aryl group, as defined herein, a monocyclicheteroaryl group, as defined herein, or a monocyclic heterocycle, asdefined herein. The tricyclic heteroaryl groups are exemplified by amonocyclic heteroaryl ring appended to the parent molecular moiety andfused to two of a monocyclic cycloalkyl group, as defined herein, amonocyclic aryl group, as defined herein, a monocyclic heteroaryl group,as defined herein, or a monocyclic heterocycle, as defined herein.Representative examples of monocyclic heteroaryl include, but are notlimited to, pyridinyl (including pyridin-2-yl, pyridin-3-yl,pyridin-4-yl), pyrimidinyl, pyrazinyl, thienyl, furyl, thiazolyl,thiadiazolyl, isoxazolyl, pyrazolyl, and 2-oxo-1,2-dihydropyridinyl.Representative examples of bicyclic heteroaryl include, but are notlimited to, chromenyl, benzothienyl, benzodioxolyl, benzotriazolyl,quinolinyl, thienopyrrolyl, thienothienyl, imidazothiazolyl,benzothiazolyl, benzofuranyl, indolyl, quinolinyl, imidazopyridine,benzooxadiazolyl, and benzopyrazolyl. Representative examples oftricyclic heteroaryl include, but are not limited to, dibenzofuranyl anddibenzothienyl. The monocyclic, bicyclic, and tricyclic heteroaryls areconnected to the parent molecular moiety through any carbon atom or anynitrogen atom contained within the rings, and can be unsubstituted orsubstituted.

The term “heterocycle” or “heterocyclic” as used herein, means amonocyclic heterocycle, a bicyclic heterocycle, or a tricyclicheterocycle. The monocyclic heterocycle is a three-, four-, five-, six-,seven-, or eight-membered ring containing at least one heteroatomindependently selected from the group consisting of O, N, and S. Thethree- or four-membered ring contains zero or one double bond, and oneheteroatom selected from the group consisting of O, N, and S. Thefive-membered ring contains zero or one double bond and one, two orthree heteroatomns selected from the group consisting of O, N and S. Thesix-membered ring contains zero, one or two double bonds and one, two,or three heteroatonms selected from the group consisting of O, N, and S.The seven- and eight-membered rings contains zero, one, two, or threedouble bonds and one, two, or three heteroatomns selected from the groupconsisting of O, N, and S. Representative examples of monocyclicheterocycles include, but are not limited to, azetidinyl, azepanyl,aziridinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl,1,3-dithianyl, 1,3-dimethylpyrimidine-2,4(1H,3H)-dione, imidazolinyl,imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl,isoxazolidinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl,oxazolidinyl, oxetanyl, piperazinyl, piperidinyl, pyranyl, pyrazolinyl,pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl,tetrahydropyranyl, tetrahydropyridinyl, tetrahydrothienyl,thiadiazolinyl, thiadiazolidinyl, 1,2-thiazinanyl, 1,3-thiazinanyl,thiazolinyl, thiazolidinyl, thiomorpholinyl, 1,1-dioxidothiomorpholinyl(thiomorpholine sulfone), thiopyranyl, and trithianyl. The bicyclicheterocycle is a monocyclic heterocycle fused to a phenyl group, or amonocyclic heterocycle fused to a monocyclic cycloalkyl, or a monocyclicheterocycle fused to a monocyclic cycloalkenyl, or a monocyclicheterocycle fused to a monocyclic heterocycle, or a spiro heterocyclegroup, or a bridged monocyclic heterocycle ring system in which twonon-adjacent atoms of the ring are linked by an alkylene bridge of 1, 2,3, or 4 carbon atoms, or an alkenylene bridge of two, three, or fourcarbon atoms. Representative examples of bicyclic heterocycles include,but are not limited to, benzopyranyl, benzothiopyranyl, chromanyl,2,3-dihydrobenzofuranyl, 2,3-dihydrobenzothienyl,2,3-dihydroisoquinoline, 2-azaspiro[3.3]heptan-2-yl,azabicyclo[2.2.1]heptyl (including 2-azabicyclo[2.2.1]hept-2-yl),2,3-dihydro-1H-indolyl, isoindolinyl, octahydrocyclopenta[c]pyrrolyl,octahydropyrrolopyridinyl, and tetrahydroisoquinolinyl. Tricyclicheterocycles are exemplified by a bicyclic heterocycle fused to a phenylgroup, or a bicyclic heterocycle fused to a monocyclic cycloalkyl, or abicyclic heterocycle fused to a monocyclic cycloalkenyl, or a bicyclicheterocycle fused to a monocyclic heterocycle, or a bicyclic heterocyclein which two non-adjacent atoms of the bicyclic ring are linked by analkylene bridge of 1, 2, 3, or 4 carbon atoms, or an alkenylene bridgeof two, three, or four carbon atoms. Examples of tricyclic heterocyclesinclude, but are not limited to, octahydro-2,5-epoxypentalene,hexahydro-2H-2,5-methanocyclopenta[b]furan,hexahydro-1H-1,4-methanocyclopenta[c]furan, aza-adamantane(1-azatricyclo[3.3.1.1³′7]decane), and oxa-adamantane(2-oxatricyclo[3.3.1.1³′7]decane). The monocyclic, bicyclic, andtricyclic heterocycles are connected to the parent molecular moietythrough any carbon atom or any nitrogen atom contained within the rings,and can be unsubstituted or substituted.

The term “hydroxyl” as used herein, means an —OH group.

In some instances, the number of carbon atoms in a hydrocarbylsubstituent (e.g., alkyl or cycloalkyl) is indicated by the prefix“C_(x)-C_(y)-”, wherein x is the minimum and y is the maximum number ofcarbon atoms in the substituent. Thus, for example, “C₁-C₃-alkyl” refersto an alkyl substituent containing from 1 to 3 carbon atoms.

The term “substituents” refers to a group “substituted” on an aryl,heteroaryl, phenyl or pyridinyl group at any atom of that group. Anyatom can be substituted.

The term “substituted” refers to a group that may be further substitutedwith one or more non-hydrogen substituent groups. Substituent groupsinclude, but are not limited to, halogen, ═O, ═S, cyano, nitro,fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl,haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl,heteroaryl, heterocycle, cycloalkylalkyl, heteroarylalkyl, arylalkyl,hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene, aryloxy, phenoxy,benzyloxy, amino, alkylamino, acylamino, aminoalkyl, arylamino,sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl,aminosulfonyl, sulfinyl, —COOH, ketone, amide, carbamate, and acyl.

For compounds described herein, groups and substituents thereof may beselected in accordance with permitted valence of the atoms and thesubstituents, such that the selections and substitutions result in astable compound, e.g., which does not spontaneously undergotransformation such as by rearrangement, cyclization, elimination, etc.

For the recitation of numeric ranges herein, each intervening numberthere between with the same degree of precision is explicitlycontemplated. For example, for the range of 6-9, the numbers 7 and 8 arecontemplated in addition to 6 and 9, and for the range 6.0-7.0, thenumber 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 areexplicitly contemplated.

2. COMPOUNDS

Disclosed are compounds of formula (I):

or tautomers, or pharmaceutically acceptable salts thereof, wherein R¹is alkyl, alkenyl, alkynyl, aryl, bicyclic aryl, tricyclic aryl,heteroaryl, bicyclic heteroaryl, tricyclic heteroaryl, heterocycle,cycloalkyl, heteroarylcarbonyl, or phosphonate; and q is 0-2; whereinsaid alkyl, alkenyl, alkynyl, aryl, bicyclic aryl, tricyclic aryl,heteroaryl, bicyclic heteroaryl, tricyclic heteroaryl, heterocycle,cycloalkyl, heteroarylcarbonyl, and phosphonate, at each occurrence, areindependently substituted or unsubstituted.

In certain embodiments, R¹ is alkyl, alkenyl, alkynyl, aryl, bicyclicaryl, tricyclic aryl, heteroaryl, bicyclic heteroaryl, tricyclicheteroaryl, heterocycle, cycloalkyl, heteroarylcarbonyl, or phosphonate;and q is 0-2; wherein said alkyl, alkenyl, alkynyl, aryl, bicyclic aryl,tricyclic aryl, heteroaryl, bicyclic heteroaryl, tricyclic heteroaryl,heterocycle, cycloalkyl, heteroarylcarbonyl, and phosphonate, at eachoccurrence, are independently substituted or unsubstituted with 1, 2, 3,4, 5, 6, or 7 functional groups independently selected from the groupconsisting halogen, ═O, ═S, cyano, carbamate, nitro, fluoroalkyl,alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl,haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl,heterocycle, heterocycloalkyl, cycloalkylalkyl, heteroarylalkyl,arylalkyl, hydroxy, hydroxyalkyl, alkoxy, allyloxy, alkoxyalkyl,alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, acylamino,aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl,alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, —COOH, ketone,amide, carbamate, silyl, substituted silyloxy, t-butyldimethylsilyloxy,alkylsulfanyl, sulfanyl, thiotriazolyl, and acyl.

In certain embodiments, R¹ is alkyl, alkenyl, alkynyl, aryl, bicyclicaryl, tricyclic aryl, heteroaryl, bicyclic heteroaryl, tricyclicheteroaryl, heterocycle, cycloalkyl, heteroarylcarbonyl, or phosphonate;and q is 0-2; wherein said alkyl, alkenyl, alkynyl, aryl, bicyclic aryl,tricyclic aryl, heteroaryl, bicyclic heteroaryl, tricyclic heteroaryl,heterocycle, cycloalkyl, heteroarylcarbonyl, and phosphonate, at eachoccurrence, are independently substituted or unsubstituted;

provided that if q is 0, then

R¹ is not C₁-C₅ alkyl or

wherein R^(a) is NH₂, halogen, OH, NHC(O)C₁-C₇-alkyl, or CO₂C₁-C₇-alkyl;

-   -   provided that if q is 1, then

R¹ is not C₁-C₄ alkyl,

wherein R^(c) is H, OH, OC(O)C₁-C₇-alkyl, OCH₂OC(O)C₁-C₇-alkyl, X is S,O, NH, NCH₃, or NCH₂CH₃, and Z is CH or N; and

provided that if q is 2, then

R¹ is not C₁-C₃ alkyl; and wherein the compound of formula (I) is not8-benzyl-2-((1-methyl-1H-imidazol-2-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one.

In certain embodiments, R¹ is alkyl, alkenyl, alkynyl, aryl, bicyclicaryl, tricyclic aryl, heteroaryl, bicyclic heteroaryl, tricyclicheteroaryl, heterocycle, cycloalkyl, heteroarylcarbonyl, or phosphonate;and q is 0-2; wherein said alkyl, alkenyl, alkynyl, aryl, bicyclic aryl,tricyclic aryl, heteroaryl, bicyclic heteroaryl, tricyclic heteroaryl,heterocycle, cycloalkyl, heteroarylcarbonyl, and phosphonate, at eachoccurrence, are independently substituted or unsubstituted with 1, 2, 3,4, 5, 6, or 7 functional groups independently selected from the groupconsisting of halogen, ═O, ═S, cyano, carbamate, nitro, fluoroalkyl,alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl,haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl,heterocycle, heterocycloalkyl, cycloalkylalkyl, heteroarylalkyl,arylalkyl, hydroxy, hydroxyalkyl, alkoxy, allyloxy, alkoxyalkyl,alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, acylamino,aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl,alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, —COOH, ketone,amide, carbamate, silyl, substituted silyloxy, t-butyldimethylsilyloxy,alkylsulfanyl, sulfanyl, thiotriazolyl, and acyl;

-   -   provided that if q is 0, then

R¹ is not C₁-C₅ alkyl or

wherein R^(a) is NH₂, halogen, OH, NHC(O)C₁-C₇-alkyl, or CO₂C₁-C₇-alkyl;

provided that if q is 1, then

R¹ is not C₁-C₄ alkyl,

or wherein R^(c) is H, OH, OC(O)C₁-C₇-alkyl, OCH₂OC(O)C₁-C₇-alkyl, X isS, O, NH, NCH₃, or NCH₂CH₃, and Z is CH or N; and

provided that if q is 2, then

R¹ is not C₁-C₃ alkyl; and wherein the compound of formula (I) is not8-benzyl-2-((1-methyl-1H-imidazol-2-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one.

In certain embodiments, R¹ is phenyl, pyridinyl, benzodioxolyl,benzotriazolyl, thiazolyl, thiadiazolyl, thienopyrrolyl, pyrimidinyl,pyrazinyl, thienothienyl, thienyl, diethylphosphonate, isoxazolyl,imidazothiazolyl, 1,3-dimethylpyrimidine-2,4(1H,3H)-dionyl, furanyl,pyrazolyl, benzothienyl, C₁₀-C₁₂ alkyl, benzothiazolyl, cinnamyl,dibenzofuranyl, chromenyl or naphthalenyl; and q is 0-2; wherein saidphenyl, pyridinyl, benzodioxolyl, benzotriazolyl, thiazolyl,thiadiazolyl, thienopyrrolyl, pyrimidinyl, pyrazinyl, thienothienyl,thienyl, diethylphosphonate, isoxazolyl, imidazothiazolyl,1,3-dimethylpyrimidine-2,4(1H,3H)-dionyl, furanyl, pyrazolyl,benzothienyl, C₁₀-C₁₂ alkyl, benzothiazolyl, cinnamyl, dibenzofuranyl,chromenyl and naphthalenyl, at each occurrence, are independentlysubstituted or unsubstituted;

provided that if q is 0, then

R¹ is not C₁-C₅ alkyl or

wherein R^(a) is NH₂, halogen, OH, NHC(O)C₁-C₇-alkyl, or CO₂C₁-C₇-alkyl;

provided that if q is 1, then

R¹ is not C₁-C₄ alkyl,

wherein R^(c) is H, OH, OC(O)C₁-C₇-alkyl, OCH₂OC(O)C₁-C₇-alkyl, X is S,O, NH, NCH₃ or NCH₂CH₃, and Z is CH or N; and

provided that if q is 2, then

R¹ is not C₁-C₃ alkyl; and wherein the compound of formula (I) is not8-benzyl-2-((1-methyl-1H-imidazol-2-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one.

In certain embodiments, R¹ is phenyl, pyridinyl, benzodioxolyl,benzotriazolyl, thiazolyl, thiadiazolyl, thienopyrrolyl, pyrimidinyl,pyrazinyl, thienothienyl, thienyl, diethylphosphonate, isoxazolyl,imidazothiazolyl, 1,3-dimethylpyrimidine-2,4(1H,3H)-dionyl, furanyl,pyrazolyl, benzothienyl, C₁₀-C₁₂ alkyl, benzothiazolyl, cinnamyl,dibenzofuranyl, chromenyl or naphthalenyl; and q is 0-2; wherein saidphenyl, pyridinyl, benzodioxolyl, benzotriazolyl, thiazolyl,thiadiazolyl, thienopyrrolyl, pyrimidinyl, pyrazinyl, thienothienyl,thienyl, diethylphosphonate, isoxazolyl, imidazothiazolyl,1,3-dimethylpyrimidine-2,4(1H,3H)-dionyl, furanyl, pyrazolyl,benzothienyl, C₁₀-C₁₂ alkyl, benzothiazolyl, cinnamyl, dibenzofuranyl,chromenyl and naphthalenyl, at each occurrence, are independentlysubstituted or unsubstituted with 1, 2, 3, 4, 5, 6, or 7 functionalgroups independently selected from the group consisting of halogen, ═O,═S, cyano, carbamate, nitro, fluoroalkyl, alkoxyfluoroalkyl,fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy,heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocycle,heterocycloalkyl, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy,hydroxyalkyl, alkoxy, allyloxy, alkoxyalkyl, alkylene, aryloxy, phenoxy,benzyloxy, amino, alkylamino, acylamino, aminoalkyl, arylamino,sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl,aminosulfonyl, sulfinyl, —COOH, ketone, amide, carbamate, silyl,substituted silyloxy, t-butyldimethylsilyloxy, alkylsulfanyl, sulfanyl,thiotriazolyl, and acyl;

provided that if q is 0, then

R¹ is not C₁-C₅ alkyl or

wherein R^(a) is NH₂, halogen, OH, NHC(O)C₁-C₇-alkyl, or CO₂C₁-C₇-alkyl;

provided that if q is 1, then

R¹ is not C₁-C₄ alkyl,

wherein R^(c) is H, OH, OC(O)C₁-C₇-alkyl, OCH₂OC(O)C₁-C₇-alkyl, X is S,O, NH, NCH₃, or NCH₂CH₃, and Z is CH or N; and

provided that if q is 2, then

R¹ is not C₁-C₃ alkyl; and wherein the compound of formula (I) is not8-benzyl-2-((1-methyl-1H-imidazol-2-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one.

Representative compounds of formula (I) include, but are not limited to:

-   8-benzyl-2-(4-fluorobenzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   8-benzyl-2-(2,4-difluorobenzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   8-benzyl-2-(3-methoxybenzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   8-benzyl-2-(4-fluoro-3-methoxybenzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   8-benzyl-2-(3-hydroxybenzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   8-benzyl-2-(4-fluoro-3-hydroxybenzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   8-benzyl-2-(3-fluoro-4-methoxybenzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   8-benzyl-2-(4-fluoro-2-methoxybenzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   8-benzyl-2-(4-methoxybenzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   tert-butyl    (3-((8-benzyl-3-oxo-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-2-yl)methyl)phenyl)carbamate;-   8-benzyl-2-((5-methoxythiophene-2-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   2-(3-aminobenzyl)-8-benzyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   8-benzyl-2-(4-(tert-butyl)benzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   8-benzyl-2-(naphthalen-2-ylmethyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one-   2-([1,1′-biphenyl]-4-ylmethyl)-8-benzyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   2-((2H-chromen-3-yl)methyl)-8-benzyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   8-benzyl-2-((1,3-diphenyl-1H-pyrazol-4-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   8-benzyl-2-(dibenzo[b,d]furan-4-ylmethyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   8-benzyl-2-cinnamyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   2-((3-acetylbenzo[d]thiazol-2(3H)-ylidene)methyl)-8-benzyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   tert-butyl    (4-((8-benzyl-3-oxo-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-2-yl)methyl)phenyl)carbamate;-   2-(4-aminobenzyl)-8-benzyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   2-(4-(allyloxy)-3-methoxybenzyl)-8-benzyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   8-benzyl-2-(4-hydroxy-3-methoxybenzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   8-benzyl-2-((5-((4-methyl-4H-1,2,4-triazol-3-yl)thio)furan-2-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   8-benzyl-6-phenyl-2-((5-(pyridine-2-yl)thiophen-2-yl)methyl)imidazo[1,2-a]pyrazin-3(7H)-one-   2-(benzo[b]thiophen-3-ylmethyl)-8-benzyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   8-benzyl-2-dodecyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   2-(benzo[b]thiophen-2-ylmethyl)-8-benzyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   8-benzyl-6-phenyl-2-(pyridin-4-ylmethyl)imidazo[1,2-a]pyrazin-3(7H)-one;-   8-benzyl-2-((5-cyclohexylthiophen-2-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   2-([2,2′-bithiophen]-5-ylmethyl)-8-benzyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   8-benzyl-2-((5-isobutylthiophen-2-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   8-benzyl-2-((5-(1-methyl-5-(trifluoromethyl)-1H-pyrazol-3-yl)thiophen-2-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   8-benzyl-6-phenyl-2-((5-(trifluoromethyl)furan-2-yl)methyl)imidazo[1,2-a]pyrazin-3(7H)-one;-   4-((8-benzyl-3-oxo-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-2-yl)methyl)benzoic    acid;-   8-benzyl-2-((5-(methoxymethyl)furan-2-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   8-benzyl-2-((perfluorophenyl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   8-benzyl-2-((1-methyl-3-(thiophen-2-yl)-1H-pyrazol-5-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   8-benzyl-2-(3,5-bis(trifluoromethyl)benzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   8-benzyl-2-((5-chloro-1-methyl-3-(trifluoromethyl)-1H-pyrazol-4-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   8-benzyl-2-((5-methylfuran-2-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   3-((8-benzyl-3-oxo-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-2-yl)methyl)benzoic    acid;-   8-benzyl-2-((5-(morpholinomethyl)furan-2-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   2-(3-(aminomethyl)benzyl)-8-benzyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   5-((8-benzyl-3-oxo-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-2-yl)methyl)-1,3-dimethylpyrimidine-2,4(1H,3H)-dione;-   8-benzyl-2-(imidazo[2,1-b]thiazol-6-ylmethyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   N-(3-((8-benzyl-3-oxo-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-2-yl)methyl)phenyl)methanesulfonamide;-   8-benzyl-2-((3,5-dimethylisoxazol-4-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   8-benzyl-6-phenyl-2-(pyridin-3-ylmethyl)imidazo[1,2-a]pyrazin-3(7H)-one;-   diethyl    (8-benzyl-3-oxo-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-2-yl)phosphonate;-   8-benzyl-2-(4-methoxy-3-(methoxymethyl)benzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   8-benzyl-2-(3-(dimethylamino)benzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   8-benzyl-2-((5-(methoxymethyl)thiophen-2-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   8-benzyl-2-(3-(methoxymethyl)benzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   8-benzyl-2-(3-(hydroxymethyl)benzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   8-benzyl-2-((2-methoxypyrimidin-5-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   8-benzyl-2-((3,4-dimethylthieno[2,3-b]thiophen-2-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   8-benzyl-6-phenyl-2-(pyrazin-2-ylmethyl)imidazo[1,2-a]pyrazin-3(7H)-one;-   8-benzyl-6-phenyl-2-((2-(propylthio)pyrimidin-5-yl)methyl)imidazo[1,2-a]pyrazin-3(7H)-one;-   8-benzyl-2-((4-methyl-4H-thieno[3,2-b]pyrrol-5-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   2-((1,2,3-thiadiazol-5-yl)methyl)-8-benzyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   8-benzyl-2-((i-methyl-1H-benzo[d][1,2,3]triazol-5-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   8-benzyl-2-(3,4-dimethoxybenzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   8-benzyl-2-(3-(methylthio)benzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   2-(benzo[d][1,3]dioxol-5-ylmethyl)-8-benzyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   8-benzyl-2-(3-((dimethylamino)methyl)benzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   8-benzyl-2-(3-ethoxybenzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   8-benzyl-2-((6-methylpyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   8-benzyl-2-((6-(dimethylamino)pyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   8-benzyl-2-((5-methoxypyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   8-benzyl-2-((6-methoxypyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   8-benzyl-2-((6-fluoropyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   8-benzyl-6-phenyl-2-(2,4,6-trifluorobenzyl)imidazo[1,2-a]pyrazin-3(7H)-one;-   8-benzyl-2-((2,6-dimethoxypyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   8-benzyl-2-((6-chloro-4-methoxypyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   8-benzyl-2-((6-fluoro-2-methoxypyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   8-benzyl-2-((2-fluoro-6-methoxypyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   8-benzyl-2-((5-fluoropyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   8-benzyl-2-((2,6-difluoropyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   8-benzyl-2-((6-bromopyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   8-benzyl-2-((6-chloropyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   8-benzyl-2-((2-fluoropyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   8-benzyl-2-((2,6-dichloropyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   8-benzyl-6-phenyl-2-styryl-1,7-dihydroimidazo[1,2-a]pyrazin-3(2H)-one;-   8-benzyl-2-(3-(2-methoxyethoxy)benzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;-   3-((8-benzyl-3-oxo-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-2-yl)methyl)benzonitrile;

and pharmaceutically acceptable salts thereof.

Compound names are assigned by using Struct=Name naming algorithm aspart of CHEMDRAW® ULTRA v. 12.0.

The compounds may exist as stereoisomers wherein asymmetric or chiralcenters are present. The stereoisomers are “R” or “S” depending on theconfiguration of substituents around the chiral carbon atom. The terms“R” and “S” used herein are configurations as defined in IUPAC 1974Recommendations for Section E, Fundamental Stereochemistry, in PureAppl. Chem., 1976, 45: 13-30. The disclosure contemplates variousstereoisomers and mixtures thereof, and these are specifically includedwithin the scope of this invention. Stereoisomers include enantiomersand diastereomers and mixtures of enantiomers or diastereomers.Individual stereoisomers of the compounds may be prepared syntheticallyfrom commercially available starting materials, which contain asymmetricor chiral centers or by preparation of racemic mixtures followed bymethods of resolution well-known to those of ordinary skill in the art.These methods of resolution are exemplified by (1) attachment of amixture of enantiomers to a chiral auxiliary, separation of theresulting mixture of diastereomers by recrystallization orchromatography, and optional liberation of the optically pure productfrom the auxiliary as described in Fumiss, Hannaford, Smith, andTatchell, “Vogel's Textbook of Practical Organic Chemistry”, 5^(th)edition (1989), Longman Scientific & Technical, Essex CM20 2JE, England,or (2) direct separation of the mixture of optical enantiomers on chiralchromatographic columns, or (3) fractional recrystallization methods.

It should be understood that the compounds may possess tautomeric forms,as well as geometric isomers, and that these also constitute an aspectof the invention.

The present disclosure also includes isotopically-labeled compounds,which are identical to those recited in formula (I), but for the factthat one or more atoms are replaced by an atom having an atomic mass ormass number different from the atomic mass or mass number usually foundin nature. Examples of isotopes suitable for inclusion in the compoundsof the invention are hydrogen, carbon, nitrogen, oxygen, phosphorus,sulfur, fluorine, and chlorine, such as, but not limited to, ²H, ³H,¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³¹P, ³²P, ³⁵S, ¹⁸F, and ³⁶Cl, respectively.Substitution with heavier isotopes such as deuterium, i.e., ²H, canafford certain therapeutic advantages resulting from greater metabolicstability, for example increased in vivo half-life or reduced dosagerequirements and, hence, may be preferred in some circumstances. Thecompound may incorporate positron-emitting isotopes for medical imagingand positron-emitting tomography (PET) studies for determining thedistribution of receptors. Suitable positron-emitting isotopes that canbe incorporated in compounds of formula (I) are ¹¹C, ¹³N, ¹⁵O, and ¹⁸F.Isotopically-labeled compounds of formula (I) can generally be preparedby conventional techniques known to those skilled in the art or byprocesses analogous to those described in the accompanying Examplesusing appropriate isotopically-labeled reagent in place ofnon-isotopically-labeled reagent.

A. Properties of the Compounds of Formula (I)

The compounds of formula (I) may be substrates of luciferases to produceluminescence. The compounds may have improved water solubility, improvedstability, improved cell permeability, increased biocompatibility withcells, reduced autoluminescence, and reduced toxicity.

“Luminescence” refers to the light output of a luciferase underappropriate conditions, e.g., in the presence of a suitable substratesuch as a coelenterazine analogue. The light output may be measured asan instantaneous or near-instantaneous measure of light output (which issometimes referred to as “T=0” luminescence or “flash”) at the start ofthe luminescence reaction, which may be initiated upon addition of thecoelenterazine substrate. The luminescence reaction in variousembodiments is carried out in a solution. In other embodiments, theluminescence reaction is carried out on a solid support. The solutionmay contain a lysate, for example from the cells in a prokaryotic oreukaryotic expression system. In other embodiments, expression occurs ina cell-free system, or the luciferase protein is secreted into anextracellular medium, such that, in the latter case, it is not necessaryto produce a lysate. In some embodiments, the reaction is started byinjecting appropriate materials, e.g., coelenterazine analogue, buffer,etc., into a reaction chamber (e.g., a well of a multiwell plate such asa 96-well plate) containing the luminescent protein. In still otherembodiments, the luciferase and/or coelenterazine analogues (e.g.,compounds of formula (I)) are introduced into a host and measurements ofluminescence are made on the host or a portion thereof, which caninclude a whole organism or cells, tissues, explants, or extractsthereof. The reaction chamber may be situated in a reading device whichcan measure the light output, e.g., using a luminometer orphotomultiplier. The light output or luminescence may also be measuredover time, for example in the same reaction chamber for a period ofseconds, minutes, hours, etc. The light output or luminescence may bereported as the average over time, the half-life of decay of signal, thesum of the signal over a period of time, or the peak output.Luminescence may be measured in Relative Light Units (RLUs).

Compounds of formula (I) can have an RLU of greater than or equal to 1,greater than or equal to 2, greater than or equal to 3, greater than orequal to 4, greater than or equal to 5, greater than or equal to 10,greater than or equal to 20, greater than or equal to 30, greater thanor equal to 40, greater than or equal to 50, or greater than or equal to100, relative to coelenterazine or a known coelenterazine analogue.

“Biocompatibility” refers to the tolerance of a cell (e.g., prokaryoticor eukaryotic) to a coelenterazine analogue (e.g., compounds of formula(I)). Biocompatibility of a coelenterazine analogue is related to thestress it causes on the host cell.

Enhanced biocompatibility of the coelenterazine analogues (e.g.,compounds of formula (I)), may be determined by measuring cell viabilityand/or growth rate of cells. For example, enhanced biocompatibility ofthe coelenterazine analogues may be determined by measuring cellviability in the absence of luciferase expression of cells exposed tothe coelenterazine analogues compared to native or known coelenterazinesto determine how compatible and/or toxic the coelenterazine analoguesare to the cells.

In particular, enhanced biocompatibility may be determined using cellviability analysis (e.g., using the CELLTITER-GLO® Luminescent CellViability assay), an apoptosis assay (e.g., using the CASPASE-GLO®technology), or another method known in the art. The effect of thecompounds of formula (I) on cell viability or apoptosis may be comparedto the effect of native or known coelenterazine analogues on cellviability or apoptosis.

Enhanced biocompatibility may also be determined by measuring the effectof the coelenterazine analogues (e.g., compounds of formula (I)) on cellgrowth or gene expression. For example, enhanced biocompatibility of thecompounds of formula (I) may be determined by measuring the cell numberafter a period of time or by determining the expression of stressresponse genes in a sample of cells that are exposed to compounds offormula (I) compared to cells exposed to a native or knowncoelenterazine or no coelenterazine. The effect of the compounds offormula (I) on cell growth or gene expression may be compared to anative or known coelenterazine.

Basic addition salts may be prepared during the final isolation andpurification of the disclosed compounds by reaction of a carboxyl groupwith a suitable base such as the hydroxide, carbonate, or bicarbonate ofa metal cation such as lithium, sodium, potassium, calcium, magnesium,or aluminum, or an organic primary, secondary, or tertiary amine.Quatemary amine salts can be prepared, such as those derived frommethylamine, dimethylamine, trimethylamine, triethylamine, diethylamine,ethylamine, tributylamine, pyridine, N,N-dimethylaniline,N-methylpiperidine, N-methylmorpholine, dicyclohexylamine, procaine,dibenzylamine, N,N-dibenzylphenethylamine, 1-ephenamine andN,N′-dibenzylethylenediamine, ethylenediamine, ethanolamine,diethanolamine, piperidine, piperazine, and the like.

B. Synthesis of Compounds of Formula (I)

Current methods that produce coelenterazine analogues employ harshreaction conditions. The limitations of these synthetic protocols haveslowed the investigation of new analogues. In addition, incompatibilityof the conditions of these methods with a variety of functional groupsand the inability to synthesize ketoaldehyde/ketoacid precursors haveled to limited structural alterations at the C-2 position ofcoelenterazine.

Herein, three robust and versatile methods that accomplish thepreparation of compounds of formula (I) are described. These methods areuseful in preparing analogues that could not be made using existingsynthetic methods. The described methodologies enable access to avariety of substituents at the C-2 position and can be performed underrelatively mild conditions, utilizing a wide variety of readilyavailable aldehydes as starting materials.

Compounds of formula (I) may be prepared by synthetic processes or bymetabolic processes. Preparation of the compounds by metabolic processesincludes those occurring in the human or animal body (in vivo) orprocesses occurring in vitro.

Compounds of formula (I), wherein the groups R¹, and q have the meaningsas set forth in the Summary of the Invention section unless otherwisenoted, can be synthesized as shown in Schemes 1-3.

Abbreviations which have been used in the descriptions of the Schemesthat follow are: Ac₂O for acetic anhydride; CDI for carbonyldiimidazole;MeOH for methanol; TMG for 1,1,3,3-tetramethylguanidine; and TFA fortrifluoroacetic acid.

As shown in Scheme 1, intermediates A-C can be prepared fromaminopyrazine i. Treatment of i with diazocarbonyls ii-iv, in thepresence of Rh₂(Oac)₄, can result in formation of aminopyrazineacetophosphonates A-C. Intermediates A-C may be stable at roomtemperature and provide starting materials for varied analogues.

Scheme 2 illustrates the conversion of intermediates A and B to thecompound of formula (I), wherein n is 1. Intermediates A and B can betreated with 1,1,3,3-tetramethylguanidine and undergoHorner-Wadsworth-Emmons olefination with aldehyde v, wherein R¹ is asdefined in the Summary of Invention, to yield intermediate vi.Intermediate vi can be reduced to give the compound of formula (I).

Scheme 3 illustrates the preparation of the compound of formula (I) bytwo routes from intermediate C. Intermediate C can be treated with1,1,3,3-tetramethylguanidine and undergo Horner-Wadsworth-Emmonsolefination with aldehyde v, wherein R¹ is as defined in the Summary ofInvention, to give intermediate vii. Intermediate vii can behydrogenated to provide intermediate viii, which can then be convertedto the compound of formula (I) by treatment with TFA and subsequentcyclization promoted by the addition of carbonyldiimidazole.Alternatively, intermediate vii can be treated with TFA, resulting inhydrolysis of the t-butyl ester and formation of intermediate ix.Addition of acetic anhydride followed by sodium borohydride can thenprovide the compound of formula (I).

Optimum reaction conditions and reaction times for each individual stepcan vary depending on the particular reactants employed and substituentspresent in the reactants used. Specific procedures are provided in theExamples section. Reactions can be worked up in the conventional manner,e.g. by eliminating the solvent from the residue and further purifiedaccording to methodologies generally known in the art such as, but notlimited to, crystallization, distillation, extraction, trituration, andchromatography. Unless otherwise described, the starting materials andreagents are either commercially available or can be prepared by oneskilled in the art from commercially available materials using methodsdescribed in the chemical literature. Starting materials, if notcommercially available, can be prepared by procedures selected fromstandard organic chemical techniques, techniques that are analogous tothe synthesis of known, structurally similar compounds, or techniquesthat are analogous to the above described schemes or the proceduresdescribed in the synthetic examples section.

Routine experimentations, including appropriate manipulation of thereaction conditions, reagents and sequence of the synthetic route,protection of any chemical functionality that cannot be compatible withthe reaction conditions, and deprotection at a suitable point in thereaction sequence of the method are included in the scope of theinvention. Suitable protecting groups and the methods for protecting anddeprotecting different substituents using such suitable protectinggroups are well known to those skilled in the art; examples of which canbe found in PGM Wuts and TW Greene, in Greene's book titled ProtectiveGroups in Organic Synthesis (4^(th) ed.), John Wiley & Sons, NY (2006),which is incorporated herein by reference in its entirety. Synthesis ofthe compounds of the invention can be accomplished by methods analogousto those described in the synthetic schemes described hereinabove and inspecific examples.

When an optically active form of a disclosed compound is required, itcan be obtained by carrying out one of the procedures described hereinusing an optically active starting material (prepared, for example, byasymmetric induction of a suitable reaction step) or by resolution of amixture of the stereoisomers of the compound or intermediates using astandard procedure (such as chromatographic separation,recrystallization or enzymatic resolution).

Similarly, when a pure geometric isomer of a compound is required, itcan be obtained by carrying out one of the above procedures using a puregeometric isomer as a starting material or by resolution of a mixture ofthe geometric isomers of the compound or intermediates using a standardprocedure such as chromatographic separation.

It can be appreciated that the synthetic schemes and specific examplesas described are illustrative and are not to be read as limiting thescope of the invention as it is defined in the appended claims. Allalternatives, modifications, and equivalents of the synthetic methodsand specific examples are included within the scope of the claims.

3. METHODS OF USE AND KITS

The compounds and proteins of the disclosure may be used in any way thatluciferase substrates, e.g., coelenterazine analogues, have been used.For example, they may be used in a bioluminogenic method which employsan analogue of coelenterazine to detect one or more molecules in asample, e.g., an enzyme, a cofactor for an enzymatic reaction, an enzymesubstrate, an enzyme inhibitor, an enzyme activator, or OH radicals, orone or more conditions, e.g., redox conditions. The sample may includean animal (e.g., a vertebrate), a plant, a fungus, physiological fluid(e.g, blood, plasma, urine, mucous secretions), a cell, a cell lysate, acell supernatant, or a purified fraction of a cell (e.g., a subcellularfraction). The presence, amount, spectral distribution, emissionkinetics, or specific activity of such a molecule may be detected orquantified. The molecule may be detected or quantified in solution,including multiphasic solutions (e.g., emulsions or suspensions), or onsolid supports (e.g., particles, capillaries, or assay vessels).

In certain embodiments, the compounds of formula (I) may be used toquantify coelenterazine. In some embodiments, a coelenterazine (e.g., anative or known coelenterazine or a compound of formula (I)) can be usedas a probe of a specific biochemical activity, e.g., apoptosis or drugmetabolism. In some embodiments, the coelenterazine concentration iscoupled to a specific enzyme activity by a “pro-coelenterazine” or“pro-substrate” that can be acted on by the specific enzyme of interest.In some embodiments, the pro-coelenterazine is a molecule that cannotsupport luminescence directly when combined with a luciferase, but canbe converted into coelenterazine through catalytic processing by aspecific enzyme of interest. In some embodiments, the approach can beused for enzymes such as those used in drug metabolism, e.g., cytochromeP450 enzymes, monoamine oxidase, and glutathione S-transferase; andapoptosis, e.g., caspases. For example, coelenterazine (e.g., a nativeor known coelenterazine, or a compound of formula (I)) can be modifiedto contain a cleavable group, such as 6′-O-methyl. In some embodiments,when incubated with a specific cytochrome P450 enzyme, the 6′O-methyl iscleaved, and the pro-coelenterazine converted to coelenterazine, whichcan be detected with a luciferase. In some embodiments, thepro-coelenterazine can be combined with other components necessary tosupport luminescence, e.g., luminescent protein such as a luciferase, toprovide a single reagent and a homogeneous assay. For example, when thereagent is added to a sample, luminescence is generated aspro-coelenterazine is converted to coelenterazine. In variousembodiments, similar assays can be developed for other enzymes, smallmolecules, or other cellular processes that can be linked to thegeneration of coelenterazines from pro-coelenterazines.

In certain embodiments, the compounds of formula (I) can be used fordetecting luminescence in live cells. In some embodiments, a luciferasecan be expressed in cells (as a reporter or otherwise), and the cellstreated with a coelenterazine (e.g., a compound of formula (I)), whichwill permeate cells in culture, react with the luciferase and generateluminescence. In addition to being cell permeant, the compounds offormula (I) show comparable biocompatibility to native coelenterazine interms of cell viability. In some embodiments, the compounds of formula(I) containing chemical modifications known to increase the stability ofnative coelenterazine in media can be synthesized and used for morerobust, live cell luciferase-based reporter assays. In still otherembodiments, a sample (including cells, tissues, animals, etc.)containing a luciferase and a compound of formula (I) may be assayedusing various microscopy and imaging techniques. In still otherembodiments, a secretable luciferase is expressed in cells as part of alive-cell reporter system.

In certain embodiments, the compounds of formula (I) disclosed hereinmay be provided as part of a kit. In some embodiments, the kit mayinclude one or more luciferases (in the form of a polypeptide, apolynucleotide, or both) and a coelenterazine, along with suitablereagents and instructions to enable a user to perform assays such asthose disclosed herein. The coelenterazine may be any of the native,known, or compounds of formula (I) disclosed herein. The kit may alsoinclude one or more buffers, such as those disclosed herein.

4. EXAMPLES Example 1. Synthesis of Common Intermediates (A, B, C)

General Procedure for the synthesis of A-C: In a 5 mL round bottom flaskwere placed aminopyrazine (1, 500 mg, 1.9 mmol), diazo compound (2-4,3.83 mmol, 2 eq.), Rh₂(OAc)₄ (84.57 mg, 10 mol %) and 3 mL ofchlorobenzene. The reaction mixture was heated at 100° C. for 24 hours.The progress of the reaction was monitored by LCMS. After 24 hours, thereaction reached 100% conversion. The mixture was adsorbed on celite andpurified on silica column using 40% EtOAc in heptane as eluent. Thedesired product was isolated pure as a brown solid with a 78-90% yield.The required diazophosphonoacetates were synthesized according to Wanget al. Synlett, 2007, 11, 1683-1686.

Methyl2-((3-benzyl-5-phenylpyrazin-2-yl)amino)-2-(diethoxyphosphoryl)acetate(A): Yield 90%; ¹H NMR (300 MHz, CDCl₃) δ=8.40 (s, 1H), 7.95-7.91 (m,2H), 7.49-7.40 (m, 2H), 7.40-7.19 (m, 6H), 5.33-5.19 (m, 2H), 4.25 (s,2H), 4.18-3.85 (m, 4H), 3.73 (s, 3H), 1.25 (t, J=6, 3H), 1.20 (t, J=6,3H); ¹³C NMR (75 MHz, cdcl3) δ=168.34, 168.32, 149.76, 149.64, 142.24,141.72, 137.14, 136.66, 136.40, 128.85, 128.77, 128.76, 128.06, 126.96,125.78, 63.72, 63.65, 63.63, 63.56, 53.55, 52.96, 51.61, 40.84, 16.33,16.29, 16.25, 16.21. ESI-MS (m/z) [M+H] (C24H29N3O5P) observed 470.

Benzyl2-((3-benzyl-5-phenylpyrazin-2-yl)amino)-2-(diethoxyphosphoryl)acetate(B): Yield 78%; ¹H NMR (300 MHz, CDCl₃) δ=8.35 (s, 1H), 7.94 (d, J=7.2,2H), 7.50-7.42 (m, 2H), 7.40-7.18 (m, 11H), 5.43-5.06 (m, 4H), 4.25 (s,2H), 4.17-3.76 (m, 4H), 1.19 (t, J=7.1, 3H), 1.16 (t, J=7.0, 3H); ¹³CNMR (75 MHz, cdcl3) δ=167.70, 167.68, 149.82, 149.70, 142.27, 141.73,137.18, 136.63, 136.39, 135.20, 128.85, 128.79, 128.77, 128.43, 128.30,128.29, 128.07, 126.94, 125.80, 67.57, 63.72, 63.64, 63.60, 63.51,53.96, 52.04, 40.84, 16.29, 16.24, 16.21, 16.16; ESI-MS (m/z) [M+H](C30H32N3O5P) observed 546.

tert-Butyl2-((3-benzyl-5-phenylpyrazin-2-yl)amino)-2-(diethoxyphosphoryl)acetate(C): Yield 83%; ¹H NMR (300 MHz, CDCl₃) δ=8.41 (s, 1H), 7.95 (d, J=5.2,2H), 7.52-7.16 (m, 8H), 5.30-5.06 (m, 2H), 4.31-4.17 (m, 2H), 4.15-3.83(m, 4H), 1.44 (s, 9H), 1.25 (t, J=9, 3H), 1.19 (t, J=9 3H); ¹³C NMR (75MHz, cdcl3) δ=166.54, 166.52, 149.89, 149.79, 141.94, 141.60, 137.25,136.58, 136.49, 128.80, 128.75, 127.98, 126.84, 125.73, 82.86, 63.46,63.38, 63.29, 63.20, 54.43, 52.50, 40.75, 27.84, 16.34, 16.26; ESI-MS(m/z) [M+H] (C27H35N3O5P) observed 512.

Example 2. Method I for the Synthesis of Compounds of Formula (I)

The development of methodology for synthesizing compounds of formula (I)was initiated by investigating reaction conditions for the HWEolefination shown in the scheme above. A variety of bases were screenedin a HWE olefination reaction between p-fluorobenzaldehyde and compoundA in THF. These initial experiments revealed that when the pKa of thebase employed is less than 11 (K₂CO₃ or DIPEA), the reaction did notproceed. However, stronger nitrogenous or alkali metal bases with pKa≥12 produced complex mixtures of decomposition products. Surprisingly,employment of 1,1,3,3-tetramethylguanidine (TMG) in THF (pKa ˜13.6) gaverise to the olefination product in low yield (see entry 2 of Table 1).

Most aldehydes, when employed in the HWE reaction with compound A, didnot exclusively provide the unsaturated ester x. Instead, x tended tocyclize to generate vi (see Scheme 5). However, these compounds weregenerally unstable, especially in non-protic solvents and at elevatedpH. Further experiments with the solvent/base combination revealed thata wide range of protic solvents stabilize the cyclization product vi,with MeOH being preferred (see scheme below and Table 1), althoughpretreatment of A with TMG in methanol led to the cyclizeddiethylphosponate [diethyl(8-benzyl-3-oxo-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-2-yl)phosphonate,compound 1].

In a model system employing p-fluorobenzaldehyde, several nitrogenousbases led to the desired cyclized product E in the reaction between Aand p-fluorobenzaldehyde. In general, there was no advantage to usingbases other than TMG in MeOH (Table 1).

SM conversion D E entry Solvent Base time % (%)* (%)*  1 DichloromethaneTMG 4 h 95 30.9 15  2 THF TMG 4 h 100 10 37  3 Toluene TMG 4 h 100 34 16 4 Acetonitrile TMG 4 h 100 9 0  5 DMF TMG 4 h 100 5 0  6 Pyridine TMG 4h 100 12 0  7 Dioxane TMG 4 h 79 43 31  8 Methanol TMG 4 h 100 0 94  9Methanol DBU 4 h 100 0 79 10 Methanol t-Bu- 4 h 100 0 97 TMG 11 MethanolTBD 4 h 100 0 77 12 Methanol MeTBD 4 h 100 0 56 13 Ethanol TMG 4 h 100 070 14 Ethylene glycol TMG 4 h 100 11 78 15 Butanol TMG 4 h 100 17 28 16isopropanol TMG 4 h 100 5 24 Acronyms of bases and solvents used in thescreening of the reaction conditions: TMG-1,1,3,3-Tetramethylguanidine;DBU-1,8-Diazabicycloundec-7-ene;t-BuTMG-2-tert-Butyl-1,1,3,3-tetramethylguanidine;TBD-1,5,7-Triazabicyclo[4.4.0]dec-5-ene;MeTBD-7-Methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene; *Yields of theproducts D and E were obtained through integration of the LCMS traces ofthe corresponding reaction mixtures. Conditions: room temperature, 10 mgof A, solvent (4 mL), TMG (3 eq.), 3 eq. 4-fluorobenzaldehyde aldehyde

It was surmised that in the reaction between A and p-fluorobenzaldehyde,the product ratio of D and E was determined by an equilibriumestablished in the TMG-methanol solution, and the instability of E underthese conditions led to a complete decay of the reaction componentswithin 24 hours. Individually isolated D and E re-exposed to thereaction conditions provided similar ratios of D and E. Furthermore,when the reaction was carried out in n-butanol, similar ratios of D andE were generated, but the n-Bu-ester (R⁵=n-Bu) also formed. Therefore,for the widest scope of substrates, methanol as solvent providedreliable access to E. Typically, synthesis of E in methanol requiredless than 4 h and proceeded in good yield if the reaction was quenchedbefore significant decomposition of E was observed.

General procedure for Method I: In a 20 mL vial was placed methyl2-((3-benzyl-5-phenylpyrazin-2-yl)amino)-2-(diethoxyphosphoryl)acetate(A) (100 mg, 0.21 mmol, 1 eq.), aldehyde (0.23 mmol, 1.1 eq.), and 12 mLof methanol. To that solution, 1,1,3,3-tetramethylguanidine (74 mg, 0.64mmol, 3 eq.) was added. The reaction mixture was stirred at roomtemperature until it reached maximum conversion (2-6 hours). Theprogress of the reaction was monitored by LCMS. The mixture was pouredinto water, extracted with ethyl acetate, and dried over MgSO₄. Thedrying agent was filtered off, and the solvent was concentrated underreduced pressure. The residue was subjected to flash chromatography onsilica gel using dichloromethane as eluent. The correspondingdehydrocoelenterazine with the general structure vi was isolated as ared solid and used in the next step without further purification.Dehydrocoelenterazine vi was dissolved in 25 mL of dichloromethane and10 mL of methanol and cooled to 0° C. To this solution, NaBH₄ (24.2 mg,0.64 mmol, 3 eq.) was added, and the reaction mixture stirred at 0° C.for 30 minutes. The reaction mixture was quenched with the 50 mL of 0.1M HCl, extracted with dichloromethane, and dried over MgSO₄. The dryingagent was filtered off, the solvent was concentrated under reducedpressure, and the residue was purified on silica gel usingdichloromethane/methanol as eluent. The target coelenterazine analoguewas isolated pure as a yellow solid and dried on high vacuum.

The following compounds were made using common intermediate A and thegeneral procedure of Method I above. Yields were calculated for the 2step process starting from intermediate A.

Yield compound R¹ (%) MS [M + H] 2

63 410 3

85 428 4

59 442 5

60 448 6

45 448 7

72 448 8

37 422 9

18 407 10

20 436 11

32 447 12

36 478 13

56 411 14

47 534 15

39 424 16

45 454 17

37 422 18

21 440 19

9 408 20

15 426 21

32 440 22

36 440 23

20 507 24

31 428 25

87 407 26

37 468 27

40 446 28

26 482 29

7 418 30

43 507 31

48 478 32

63 438 33

7 495 34

13 475 35

18 470 36

4 393 37

5 480 38

6 480 39

11 454 40

7 546 41

11 450 42

31 426 43

10 482 44

10 528 45

5 498 46

13 396 47

43 436 48

29 421 49

8 485 50

21 466 51

29 435 52

27 442 53

27 436 54

27 422 55

7 482 56

36 468 57

25 452 58

37 438 59

24 436 60

19 449 61

45 436 62

30 466

Example 3. Modification of Method I for the Synthesis of Compounds ofFormula (I)

Further investigation of Method I showed that the use of intermediate Bwas advantageous in cases where the equilibrium between the cyclized anduncyclized olefin intermediate is slow. After the olefination reactionwas complete, vi quickly formed, and the uncyclized intermediate was notdetected in the reaction mixture. However, vi did undergo ring openingby methoxide attack, affording x. To avoid formation of x, the reactionwas halted after 100% conversion of B (10-20 minutes). Reduction of viafforded the compound of formula (I) smoothly, though. This two-stepprocess can be completed within 10-20 minutes. Moreover, yields ofchallenging substrates can be increased.

Throughout these studies it was observed that electron withdrawingsubstituents at the R¹ position tend to favor the formation of closedform vi. This was taken advantage of and modified Method I was used toprepare compounds of formula (I) containing pyridinyl groups at the R¹position.

The following compounds were made in an analogous fashion using commonintermediate B and the general procedure of Method I in Example 2.Yields were calculated for the 2 step process starting from intermediateB.

Yield MS compound R¹ (%) [M + H] 63

33 441 64

13.7 393 65

27.9 429 66

35.9 411 67

29.2 411 68

16 411 69

28 427 70

25.4 471 71

72.1 423 72

34.8 423 73

42 453 74

32.2 457 75

25.9 461 76

35.1 407 77

27.5 436

Example 4. Methods II and III for the Synthesis of Compounds of Formula(I)

Method I is a convenient and fast method with short reaction times thatenables access to a variety of compounds of formula (I). However, manycompounds of formula (I) containing electron deficient aromatic rings orreactive heterocyclic rings at the R¹ position could not be made byMethod I. In some cases, olefination and subsequent cyclization wereslow, and significant decomposition of the products was observed. On theother hand, electron rich aldehydes tended to have low conversion ratesin the HWE olefination reaction which led to the formation ofsignificant amounts of 1, an unproductive intermediate (see Example 2).

To circumvent these challenges, two alternate routes were developed.Utilization of C prevented cyclization to the imidazolinone ring duringthe HWE reaction, providing only unsaturated ester vii. Subsequenttreatment with TFA generated unsaturated acid ix, which was converted tothe compound of formula (I) through a two-step cyclization and reductionsequence (Method II). In this method, vii was typically isolated inexcellent yield. Following the hydrolysis approach, Method II avoidsexposing sensitive intermediates to high pH conditions. Thus, in caseswhen the product ix is stable to TFA treatment, Method II affordedcompounds of formula (I) with higher yields than Method I, although theprocess was lengthier.

General procedure for Method II: In a 20 mL vial was placed tert-butyl2-((3-benzyl-5-phenylpyrazin-2-yl)amino)-2-(diethoxyphosphoryl)acetate(C) (200 mg, 0.39 mmol, 1 eq.), aldehyde (0.43 mmol, 1.1 eq.), and 15 mLof methanol. To that solution, 1,1,3,3-tetramethylguanidine (135 mg, 1.2mmol, 3 eq.) was added, and the reaction mixture was stirred at roomtemperature for 2 hours. The progress of the reaction was monitored byLCMS. The reaction mixture was poured into water extracted with ethylacetate and dried over MgSO₄. The drying agent was filtered off, thesolvent was concentrated under reduced pressure, and the product withthe general structure vii was used in the next step without furtherpurification.

To a solution of vii in dichloromethane (5 mL), trifluoroacetic acid (2mL) was added. The reaction mixture stirred at room temperature for 4hours. The progress of the reaction was monitored by LCMS. After thereaction was complete, all volatiles were removed under reducedpressure, and the residue was dried under high vacuum. The resultingunsaturated acid was dissolved in THF (10 mL), and to this solution,acetic anhydride (398 mg, 3.9 mmol, 10 eq.), triethylamine (395 mg, 3.9mmol, 10 eq.) and DMAP (4.8 mg, 0.039 mmol, 0.1 eq.) were added. Thereaction mixture stirred at room temperature for 30 min and poured inthe water, then extracted with dichloromethane, and dried over MgSO₄.The drying agent was filtered off, and the solvent was concentratedunder reduced pressure. The residue was subjected to a flashchromatography on silica gel using dichloromethane as eluent. Thecorresponding dehydrocoelenterazine with the general structure vi wasisolated as red solid and used in the next step without furtherpurification.

Dehydrocoelenterazine vi was dissolved in 25 mL of dichloromethane and10 mL of methanol and cooled to 0° C. To this solution, NaBH₄ (44.26 mg,1.2 mmol, 3 eq.) was added, and the reaction mixture stirred at 0° C.for 30 minutes. The reaction mixture was quenched with 100 mL of 0.1 MHCl, extracted with dichloromethane, and dried over MgSO₄. The dryingagent was filtered off, the solvent was concentrated under reducedpressure, and the residue was purified on silica gel usingdichloromethane/methanol as eluent. The target coelenterazine analogueof formula (I) was isolated pure as a yellow solid and dried on highvacuum.

Method III is a second alternative that bypasses all potentiallyunstable synthetic intermediates. Hydrogenation of unsaturated estervii, which was achieved in the presence of Wilkinson's catalyst underelevated pressure of hydrogen, yielded intermediate viii, which wastypically easy to isolate and purify. TFA treatment of viii, followed byintramolecular cyclization of the corresponding acid using carbonyldiimidazole (CDI) afforded the compound of formula (I). Method IIIemploys mild reaction conditions and avoids oxygen and moisturesensitive reaction intermediates; it is suitable both for scale-up andpreparation of coelenterazine analogue containing sensitive functionalgroups. However, high pressure and prolonged reaction times arerequired.

General procedure for Method III: In a 20 mL vial was placed tert-butyl2-((3-benzyl-5-phenylpyrazin-2-yl)amino)-2-(diethoxyphosphoryl)acetate(C) (200 mg, 0.39 mmol, 1 eq.), aldehyde (0.43 mmol, 1.1 eq.), and 15 mLof methanol. To that solution, 1,1,3,3-tetramethylguanidine (135 mg, 1.2mmol, 3 eq.) was added, and the reaction mixture was stirred at roomtemperature for 1-2 hours. The progress of the reaction was monitored byLCMS. After the reaction was complete, the mixture was poured intowater, extracted with ethyl acetate, and dried over MgSO₄. The dryingagent was filtered off, and the solvent was concentrated under reducedpressure. The residue was purified using flash chromatography on silicagel with heptanelethyl acetate as eluent and target compound vii wasisolated as colorless oil.

In a parr-shaker reactor flask was placed tert-butyl2-(3-benzyl-5-phenylpyrazin-2-ylamino)-acrylate derivative vii (0.3mmol, 1 eq.), Rh(PPh₃)₃Cl (0.03 mmol, 0.1 eq.), and 50 mL of ethanol.The reactor was charged with H2 (50 psi) and shaken for 20 hours at roomtemperature. The reaction was monitored by LCMS. Then, all volatileswere removed under reduced pressure, and the residue was subjected toflash chromatography on silica gel using heptane/ethylacetate as eluent.The reduced aminopirazine derivative viii was isolated pure as colorlessoil.

Compound viii (0.25 mmol, 1 eq.) was dissolved in dichloromethane (5mL), and to this solution, trifluoroacetic acid (2 mL) was added. Thereaction mixture stirred at room temperature for 12 hours. The progressof the reaction was monitored by LCMS. After the reaction was complete,all volatiles were removed under reduced pressure, and the residue wasco-evaporated 3 times with 10 mL of toluene to remove all TFA andprovide compound xi. After drying under high vacuum, compound xi wasdissolved in dichloromethane (15 mL), and to this solution was addedcarbodiimidazole (CDI) (122 mg, 0.75 mmol, 3 eq.). The reaction mixturestirred at room temperature for 20 minutes and then poured into 0.01 MHCl (50 mL), extracted with dichloromethane, and dried over MgSO₄. Thedrying agent was filtered off, and the solvent was concentrated underreduced pressure. The residue was subjected to a flash chromatography onsilica gel using dichloromethane/methanol as eluent. The correspondingcoelenterazine of formula (I) was isolated pure as yellow solid.

The following exemplary intermediates were isolated and characterized inthe employment of Method III:

tert-Butyl(E)-2-((3-benzyl-5-phenylpyrazin-2-yl)amino)-3-(3-cyanophenyl)acrylate

¹H NMR (300 MHz, CDCl₃) δ=10.03 (s, 1H), 8.31 (s, 1H), 8.15 (td, J=0.6,1.6, 1H), 8.12-8.08 (m, 1H), 7.96-7.87 (m, 2H), 7.68 (t, J=7.7, 1H),7.51-7.35 (m, 4H), 7.33-7.23 (m, 3H), 7.21-7.07 (m, 1H), 6.98 (s, 1H),6.34 (s, 1H), 4.30 (s, 2H), 1.46 (s, 9H); ¹³C NMR (75 MHz, CDCl₃)δ=189.91, 164.29, 148.16, 143.74, 142.89, 137.18, 136.85, 136.79,136.76, 136.62, 136.13, 133.27, 133.12, 132.29, 132.23, 131.08, 130.23,130.09, 129.20, 129.12, 128.83, 128.54, 128.49, 127.41, 126.02, 121.99,118.39, 117.54, 113.68, 112.55, 104.99, 82.43, 41.15, 27.89; ESI-MS(m/z) [M+H] (C31H29N4O2) observed 489.

tert-Butyl2-((3-benzyl-5-phenylpyrazin-2-yl)amino)-3-(3-cyanophenyl)propanoate

¹H NMR (300 MHz, CDCl₃) δ=8.40 (s, 1H), 7.98-7.92 (m, 2H), 7.50-7.43 (m,3H), 7.38-7.23 (m, 7H), 7.22-7.18 (m, 1H), 7.11 (dt, J=1.5, 7.8, 1H),5.02 (d, J=6.8, 1H), 4.93-4.79 (m, 1H), 4.15 (q, J=15.3, 3H), 3.10 (ddd,J=5.8, 13.9, 34.0, 2H), 1.38 (s, 9H); ¹³C NMR (75 MHz, CDCl₃)=170.81,150.01, 141.21, 141.14, 138.34, 137.35, 136.79, 136.45, 133.91, 132.94,130.36, 128.93, 128.91, 128.77, 128.58, 127.88, 127.03, 125.66, 118.72,112.15, 82.50, 55.09, 40.94, 37.46, 27.96; ESI-MS (m/z) [M+H](C31H31N4O2) observed 491.

tert-Butyl(E)-2-((3-benzyl-5-phenylpyrazin-2-yl)amino)-3-(1,2,3-thiadiazol-5-yl)acrylate

¹H NMR (300 MHz, CDCl₃) δ=9.45 (s, 1H), 8.51 (s, 1H), 8.27 (s, 1H),8.03-7.96 (m, 2H), 7.52-7.45 (m, 4H), 7.43-7.31 (m, 3H), 7.30-7.22 (m,1H), 6.70 (s, 1H), 4.43 (s, 2H), 1.48 (s, 9H); ¹³C NMR (75 MHz, CDCl₃)δ=164.35, 159.03, 147.85, 144.44, 143.61, 136.90, 136.87, 136.42,135.80, 133.05, 129.07, 128.85, 128.74, 128.54, 126.81, 126.09, 104.18,82.10, 40.34, 27.87; ESI-MS (m/z) [M+H] (C₂₆H26N5O2S) observed 472

tert-Butyl2-((3-benzyl-5-phenylpyrazin-2-yl)amino)-3-(1,2,3-thiadiazol-5-yl)propanoate

¹H NMR (300 MHz, CDCl₃) δ=8.42 (s, 1H), 7.99-7.93 (m, 2H), 7.51-7.42 (m,3H), 7.41-7.23 (m, 6H), 5.41 (d, J=6.6, 1H), 5.00 (dt, J=5.0, 6.5, 1H),4.19 (dd, J=15.2, 58.4, 2H), 3.82-3.62 (m, 2H), 1.36 (s, 9H); ¹³C NMR(75 MHz, CDCl₃) δ=170.52, 158.41, 149.87, 141.26, 141.12, 137.38,136.91, 136.87, 132.95, 129.02, 128.81, 128.78, 127.88, 127.01, 125.65,82.60, 53.92, 40.98, 30.06, 27.83; ESI-MS (m/z) [M+H] (C26H28N5O2S)observed 474.

The following compounds were made by Methods II and III described above.Yields we calculated for the 4 step process starting from intermediateC.

Com- Meth- Yield MS pound R¹ od (%) [M + H] 78

II 78% 446 79

III 30% 417 80

II 32% 438 81

II 25% 481 82

III 17% 400  83^(a)

II  6 491 84

II 12 394 85

II  7 451  86^(a)

II 10 404 ^(a)compounds were made by Method II, but excluding the finalreduction step with NaBH₄

Thus, having established multiple synthetic methodologies, the scope andutility of these methods were demonstrated by synthesizing compounds offormula (I) with a wide variety of substituents at the R¹ position. Asshown above, aromatic groups with electron-withdrawing substituents,aromatic groups with electron-donating substituents, stericallydemanding groups, and polar heterocyclic motifs were all amenable tosynthesis using one of these new methods.

Example 5. Luminescent Properties

Luminescence Assay Procedure: Each compound to be screened was dissolvedin DMSO (5 mM) and then further diluted to 100 uM in NANO-GLO®Luciferase Assay Buffer. Each diluted substrate was then combined inequal volumes with purified NanoLuc® Luciferase diluted into CO₂independent media+10% FBS. Initial light output for each substrate wasmeasured in a GloMax®-Multi+ luminometer three minutes after substrateaddition and then at five minute intervals as a means to determinesignal half-life.

A subset of the synthesized coelenterazine analogues (compounds offormula (I)) were evaluated for their suitability as luciferasesubstrates. NANOLUC® luciferase was employed for the screening becauseit is a small (19 kDa), stable, and particularly bright enzyme. Table 2demonstrates that relative light unit (RLUs) and half-life data (ratiosin comparison to native coelenterazine) indicated that moderatelyelectron-donating and electron-withdrawing functionalities were welltolerated. A variety of pyridinyl groups were also tolerated with asignificant number demonstrating superior performance to coelenterazine.Overall, a significant number of analogues were superior tocoelenterazine in generating luminescence.

TABLE 2 Compound I Half-life coelenterazine 1 1 1 — — 2 40 0.16 3 38.750.23 4 9.166 0.72 5 3.875 0.43 6 13.75 0.33 7 4.166 0.52 8 23.333 0.38 91.041 0.93 10 0.142 1.02 11 0.3 1.04 12 0.192 0.57 13 0.583 2.64 140.042 2.15 15 0.583 0.88 16 0.0042 0.005 64 2.75 3.03 65 10.833 0.62 661.625 0.98 67 4.583 1.14 68 12.916 0.93 69 7.5 0.35 70 5 0.19 71 27.0830.14 72 1.208 0.88 73 6.666 0.93 74 3 1.09 75 0.958 0.57 76 1.666 1.1477 1.75 0.57 78 8.75 0.43 79 13.87 0.52 80 2.166 1.62 81 0.246 1.19 825.416 1.19

The compounds of formula (I) were also evaluated for their ability to beluciferase substrates in an additional assay. In this assay, theanalogues were compared to a known coelenterazine analogue,8-benzyl-2-(furan-2-ylmethyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one(C-1), which is known to be superior to coelenterazine as a luciferasesubstrate. Table 3 demonstrates that a significant number of theanalogues were well tolerated and performed similarly to or better than8-benzyl-2-(furan-2-ylmethyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one.

TABLE 3 RLU half-life Compound (@100 uM) (@100 uM) Km C-1 1 1 1  10.00048 0.4 3  2 0.96 0.32 2.68  3 0.93 0.45 2.47  4 0.22 1.4 4.2  50.093 0.83 6.4  6 0.33 0.646 3.7  7 0.1 1 3  8 0.56 0.73 1.83  9 0.0251.8 5.57 10 0.0034 1.96 7.4 11 0.0072 2 3.3 12 0.0046 1.1 3.4 13 0.0145.1 4.96 14 0.001 14 15 0.014 1.7 4.3 16 0.0001 0.01 3.7 17 0.83 1.22.28 18 0.4 1.28 4.05 19 0.1 3.34 3.63 20 0.29 (75) 1.87 (75) 4.95 210.32 0.81 2.26 22  0.3 (75) 1.79 (75) 2.32 23 0.001 5 4.61 24 0.68 0.551.98 25 0.15 3.22 4.29 26 0.046 1.09 4.5 27 0.1 1.19 2.2 28 0.01 2.47 290.13 14.3 30 0.001 3.5 4.62 31 0.054 1.56 4.44 32 0.052 1.35 6.5 330.017 1.9 8.3 34 0.02 1.2 1.89 35 0.0005 4.92 7.5 36 0.0017 N/A 0.97 370.05 0.45 1.25 38 0.054 0.6 1.25 39 0.29 0.92 6.8 40 0.0042 0.71 6.2 410.82 0.6 2 42 0.52 1.3 4.5 43 0.052 0.56 3.2 44 0.019 1.1 8.2 45 0.00122.4 7.3 46 47 0.013 2.9 4 48 0.0013 4.4 11.7 49 0.002 4.4 4.8 50 0.0232.2 4 51 0.49 1.2 3.2 52 0.45 1 2.2 53 0.47 1.6 2.1 54 0.04 2.8 3.8 550.0063 1.4 4.4 56 0.0098 0.9 6.1 57 0.063 1.7 4.5 58 0.47 1.2 3.2 590.72 0.53 1.7 60 0.00038 4.1 6.9 61 0.49 1.7 3.4 62 0.057 3.36 3.97 630.28 0.5 1.6 64 0.066 5.85 1.82 65 0.26 1.2 7.1 66 0.039 1.9 2.8 67 0.112.2 6.7 68 0.31 1.8 11 69 0.18 0.68 3.6 70 0.12 0.37 3.1 71 0.65 0.281.9 72 0.029 1.7 3 73 0.16 1.8 2.7 74 0.072 2.1 3.4 75 0.023 1.1 6.5 760.04 2.2 6.3 77 0.042 1.1 5.8 78 0.21 0.83 3.8 79 0.33 4.06 80 0.0523.12 7.1 81 0.0059 2.3 6.7 82 0.13 2.3 10 83 0.02 11.1 84 0.003 3.1 8 850.038 0.5 NA 86 0.0002 NA NA

Example 6. Cell Permeability and Bioluminescent Activity

Cell Culture: HeLa and HEK293 cells were maintained in DMEM containing0.3 mg/ml glutamine, 100 IU/ml penicillin, 100 μg/ml streptomycin, and10% fetal calf serum at 37° C. in 5% CO₂. Dulbecco's modified eaglemedium (DMEM), Opti-MEM, Penicillin/Streptomycin, and Trypsin-EDTA werepurchased from Life Technologies (Carlsbad). Fetal calf serum (FBS) waspurchased from HyClone (GE Healthcare). Microtiter plates were purchasedfrom Corning.

Cell based luciferase assay: HEK293 cells stably expressing NANOLUC®luciferase under the control of a CMV promotor were plated in 100 μlgrowth medium (DMEM supplemented with 10% FBS) into wells of white,TC-treated, 96-well plates at a density of 10000 cells per well andincubated for 24 h. The growth medium was then replaced with 100 μlOptiMEM containing 12.5 μM of the indicated substrate. The luminescentsignal was analyzed immediately following substrate addition using aGLOMAX® Discover multimode detection plate reader (Promega).

Cell permeability and bioluminescent activity of certain compounds(64-77) were determined in comparison to coelenterazine using HEK293cells stably expressing NANOLUC® luciferase (FIG. 1). A majority of thecompounds tested exhibited a luminescent signal equal or stronger thanwith coelenterazine, and at least six showed a substantial increase(>5-fold) over coelenterazine. Furthermore, rank ordering compounds bysignal strength relative to coelenterazine showed a strong correlationbetween the biochemical and cell-based luciferase assay (FIG. 2).Together, these results imply that the tested analogues were freelypermeable across biological membranes and show good bioluminescentactivity within living cells.

Example 7. Cell Viability

Cell viability assay: HEK293 or HeLa cells were plated in 100 μl growthmedium (DMEM supplemented with 10% FBS) into wells of white, TC-treated,96-well plates at a density of 10000 cells per well and incubated for 24h. The growth medium was then replaced with 100 μl Opti-MEM medium,which contained a serial dilution of the indicated compound. Changes incell viability were then measured after incubation for 24 h using theCELLTITER@ Green cell viability assay (Promega) according tomanufacturer instructions. All luminescent measurements were performedon a GLOMAX® Discover multimode plate reader (Promega).

Evaluation of compound-induced cellular toxicity in HeLa (FIGS. 3 and 4)and Hek293 (FIGS. 5 and 6) cells showed no fundamental difference in thesubstrate toxicity pattern throughout the two different cell lines.Interestingly, fluorinated compounds 65-68 generally demonstratedreduced toxicity in both cell lines when compared to coelenterazine. Inaddition compound 76 also exhibited notably reduced toxicity in bothcell lines, which might enable the use of higher substrateconcentrations in cell based assays.

It is understood that the foregoing detailed description andaccompanying examples are merely illustrative and are not to be taken aslimitations upon the scope of the invention, which is defined solely bythe appended claims and their equivalents.

Various changes and modifications to the disclosed embodiments will beapparent to those skilled in the art. Such changes and modifications,including without limitation those relating to the chemical structures,substituents, derivatives, intermediates, syntheses, compositions,formulations, or methods of use of the invention, may be made withoutdeparting from the spirit and scope thereof.

What is claimed is:
 1. A compound of formula (I):

or a pharmaceutically acceptable salt or tautomer thereof, wherein: R¹is heterocyclyl or heteroaryl; and q is 1; wherein the heterocyclyl orheteroaryl is optionally substituted with 1, 2, 3, 4, 5, 6, or 7substituents independently selected from the group consisting ofhalogen, oxo, thioxo, nitro, cyano, hydroxy, alkyl, haloalkyl,hydroxyalkyl, alkoxyalkyl, aminoalkyl, alkoxyfluoroalkyl,cycloalkylalkyl, heterocyclylalkyl, arylalkyl, heteroarylalkyl, alkenyl,alkynyl, alkoxy, haloalkoxy, allyloxy, benzyloxy, aryloxy, silyloxy,sulfanyl, alkylsulfanyl, amino, alkylamino, acylamino, arylamino,sulfinylamino, sulfonylamino, acyl, carboxy, carbamate, amido, sulfinyl,sulfonyl, silyl, cycloalkyl, cycloalkenyl, heterocyclyl, aryl, andheteroaryl: with the proviso that: R¹ is not selected from the groupconsisting of:

wherein: X is NH, N(CH₃), N(CH₂CH₃), S, or O; and Z is CH or N.
 2. Thecompound of claim 1, or a pharmaceutically acceptable salt or tautomerthereof, wherein R¹ is heteroaryl: wherein the heteroaryl is optionallysubstituted with 1, 2, or 3 substituents independently selected from thegroup consisting of halogen, C₁-C₃ alkyl, haloalkyl, alkoxyalkyl,alkoxy, haloalkoxy, amino, alkylamino, cycloalkyl, heterocyclylalkyl,aryl, and heteroayl.
 3. The compound of claim 1, or a pharmaceuticallyacceptable salt or tautomer thereof, wherein R¹ is a monocyclicheteroayl.
 4. The compound of claim 1, or a pharmaceutically acceptablesalt or tautomer thereof, wherein R¹ is a bicyclic heteroaryl or atricyclic heteroaryl.
 5. The compound of claim 1, or a pharmaceuticallyacceptable salt or tautomer thereof, wherein R¹ is pyridinyl,benzodioxolyl, benzotriazolyl, thiazolyl, thiadiazolyl, thienopyrrolyl,pyrimidinyl, pyrazinyl, thienothienyl, thienyl, isoxazolyl,imidazothiazolyl, 1,3-dimethylpyrimidine-2,4(1H,3H)-dionyl, furanyl,pyrazolyl, benzothienyl, benzothiazolyl, dibenzofuranyl, or chromenyl.6. A kit comprising a compound of claim 1, or a pharmaceuticallyacceptable salt or tautomer thereof.
 7. The kit of claim 6, wherein thekit further comprises a luciferase.
 8. The kit of claim 6, wherein thekit further comprises a buffer reagent.
 9. A method for detectingluminescence in a sample, the method comprising: contacting a samplewith a compound of claim 1, or a pharmaceutically acceptable salt ortautomer thereof: contacting the sample with a coelenterazine-utilizingluciferase, if it is not present in the sample; and detectingluminescence.
 10. The method of claim 9, wherein the sample containslive cells.
 11. The method of claim 9, wherein the sample contains acoelenterazine-utilizing luciferase.
 12. A method for detectingluminescence in a transgenic animal, the method comprising:administering a compound of claim 1, or a pharmaceutically acceptablesalt or tautomer thereof, to a transgenic animal; and detectingluminescence: wherein the transgenic animal expresses acoelenterazine-utilizing luciferase.
 13. A compound selected from thegroup consisting of:8-benzyl-2-((5-methoxythiophen-2-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;2-((2H-chromen-3-yl)methyl)-8-benzyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;8-benzyl-2-((1,3-diphenyl-1H-pyrazol-4-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;8-benzyl-2-(dibenzo[b,d]furan-4-ylmethyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;8-benzyl-2-((5-((4-methyl-4H-1,2,4-triazol-3-yl)thio)furan-2-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one; 8-benzyl-6-phenyl-2-((5-(pyridin-2-yl)thiophen-2-yl)methyl)imidazo[1,2-a]pyrazin-3 (7H)-one;2-(benzo[b]thiophen-3-ylmethyl)-8-benzyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;2-(benzo[b]thiophen-2-ylmethyl)-8-benzyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;8-benzyl-6-phenyl-2-(pyridin-4-ylmethyl) imidazo[1,2-a]pyrazin-3(7H)-one;8-benzyl-2-((5-cyclohexylthiophen-2-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;2-([2,2′-bithiophen]-5-ylmethyl)-8-benzyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;8-benzyl-2-((5-isobutylthiophen-2-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;8-benzyl-2-((5-(1-methyl-5-(trifluoromethyl)-1H-pyrazol-3-yl)thiophen-2-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;8-benzyl-6-phenyl-2-((5-(trifluoromethyl)furan-2-yl)methyl)imidazo[1,2-a]pyrazin-3(7H)-one;8-benzyl-2-((5-(methoxymethyl)furan-2-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3 (7H)-one;8-benzyl-2-((1-methyl-3-(thiophen-2-yl)-1H-pyrazol-5-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;8-benzyl-2-((5-chloro-1-methyl-3-(trifluoromethyl)-1H-pyrazol-4-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;8-benzyl-2-((5-methylfuran-2-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one; 8-benzyl-2-((5-(morpholinomethyl)furan-2-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;5-((8-benzyl-3-oxo-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-2-yl)methyl)-1,3-dimethylpyrimidine-2,4(1H,3H)-dione;8-benzyl-2-(imidazo[2,1-b]thiazol-6-ylmethyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;8-benzyl-2-((3,5-dimethylisoxazol-4-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;8-benzyl-6-phenyl-2-(pyridin-3-ylmethyl)imidazo[1,2-a]pyrazin-3(7H)-one;8-benzyl-2-((5-(methoxymethyl)thiophen-2-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;8-benzyl-2-((2-methoxypyrimidin-5-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;8-benzyl-2-((3,4-dimethylthieno[2,3-b]thiophen-2-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;8-benzyl-6-phenyl-2-(pyrazin-2-ylmethyl)imidazo[1,2-a]pyrazin-3(7H)-one;8-benzyl-6-phenyl-2-((2-(propylthio)pyrimidin-5-yl)methyl)imidazo[1,2-a]pyrazin-3(7H)-one;8-benzyl-2-((4-methyl-4H-thieno[3,2-b]pyrrol-5-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;2-((1,2,3-thiadiazol-5-yl)methyl)-8-benzyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;8-benzyl-2-((1-methyl-1H-benzo[d][1,2,3]triazol-5-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;2-(benzo[d][1,3]dioxol-5-ylmethyl)-8-benzyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;8-benzyl-2-((6-methylpyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;8-benzyl-2-((6-(dimethylamino)pyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;8-benzyl-2-((5-methoxypyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;8-benzyl-2-((6-methoxypyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;8-benzyl-2-((6-fluoropyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;8-benzyl-2-((2,6-dimethoxypyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;8-benzyl-2-((6-chloro-4-methoxypyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;8-benzyl-2-((6-fluoro-2-methoxypyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;8-benzyl-2-((2-fluoro-6-methoxypyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;8-benzyl-2-((5-fluoropyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;8-benzyl-2-((2,6-difluoropyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;8-benzyl-2-((6-bromopyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;8-benzyl-2-((6-chloropyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;8-benzyl-2-((2-fluoropyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;and8-benzyl-2-((2,6-dichloropyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;or a pharmaceutically acceptable salt or tautomer thereof.